An enzyme exhibiting fructan hydrolase activity

ABSTRACT

The present invention is related to an enzyme that allows efficient removal of fructan from grain and vegetable raw material. The enzyme according to the invention produces grain and vegetable material having a fructan content significantly lower compared to that of the starting material.

FIELD OF THE INVENTION

The present invention is directed to an enzyme that allows efficientremoval of fructan from grain and vegetable raw material. The enzymeaccording to the invention produces grain and vegetable material havinga fructan content significantly lower compared to that of the startingmaterial. The low-fructan grain and vegetable materials can be used inproducing low-fructan grain and vegetable ingredients, products suitablee.g. for low -FODMAP diet, and various cereal and vegetable foodproducts with dietary benefits. The present invention is also directedto products containing low-fructan grain or vegetable ingredients and toproducts containing the enzyme, such as improvers and premixes forbaking purposes.

BACKGROUND OF THE INVENTION

Digestion-related problems are a frequent cause of general and socialdiscomfort. These problems cover a diverse selection of gastrointestinalsymptoms of which bloating, gas production, abdominal pain, overalldiscomfort, constipation, and loose stools are among the most frequent.Today many of the sufferers of such symptoms are believed to suffer fromirritable bowel syndrome (IBS). IBS is clearly more frequent in womenand it is believed to concern 10-20 % of Western population; i.e. IBS ismore frequent in Western population than lactose-intolerance (manypeople having lactose intolerance, though, might have IBS and viceversa).

Currently there is no good medical cure for IBS. Much attention has beenpaid on dietary management of IBS. Most attention has been paid on adiet called LOW-FODMAP diet. The idea of the diet is to avoid food itemsthat contain FODMAP compounds. Term FODMAP is derived from “Fermentable,Oligo-, Di-, Monosaccharides, and Polyols”. FODMAPs are short chaincarbohydrates and monosaccharides, which are poorly absorbed in thesmall intestine. FODMAP compounds include fructans (including FOS),galactans (especially GOS), and polyols. Also lactose and excessfructose can be considered as FODMAP compounds among people withimpaired digestion or absorption of these compounds.

Common sources of fructans include for example wheat, rye, onion,Jerusalem artichoke, and garlic. Some examples of fructan contents ofgrains are as follows: rye (bran) 7 % (on grain material basis), rye(grain) 3-7 %, and wheat flour 1-4 %. Although wheat is not generallyconsidered as being especially rich in FODMAP compounds, its relativelyhigh consumption makes it a relevant source of fructans. This is why theFODMAP diet guidelines instruct to avoid wheat. Rye consumption is highin Northern Europe. Rye bread contains more FODMAP compounds compared towheat bread, because whole grain rye contains more fructans than wheatflour.

Fructans are built up of fructose residues, normally with a terminalsucrose unit (i.e. a glucose -fructose disaccharide). The linkageposition of the fructose residues determines the type of the fructan.The basic types of single-linkage fructans are inulin and levan (orphlein). Additionally, there exists a mixed-linkage fructan calledgraminan.

Some prior art related to levels of fructan in bread is existing. In thearticle by Andersson et al. (2009) it was shown that the yeast fermentedbread and especially the sourdough bread had lower contents of fructanas compared to whole grain rye flour. The results of Andersson et al.show that the fructan content of whole grain rye can be reduced from5.0% to 1.9% by sourdough (62% reduction) and to 3.4% by yeastfermentation (32% reduction). The results also show that fructans aredegraded during the bread-making process resulting in lower contents oftotal and extractable dietary fiber in the bread.

Article by Rakha et al. (2010) discloses that during bread making, thelow-molecular weight fraction of fructan is most available fordegradation by yeast or by endogenous enzymes present in theingredients. According to Rakha et al., the fructan content in ryemilling fractions ranges from 3.4% in inner endosperm to 5.0% in bran.The fructan content of rye breads varied from 1.9% to 4.0%, with anaverage of 2.8% in crisp breads, with a sample containing only wholegrain rye flour being the highest in fructan content.

The dough according to US patent application US 2011/0129572 A1comprises at least one fructose-containing polysaccharide and at leastone enzyme capable of degrading said polysaccharide into short-chainedfructo-oligosaccharide (FOS) and fructose. The baked product producedusing this dough was said to have an increased softness compared tootherwise identical control bread or baked product produced using doughnot containing the enzyme.

The discovery related to lowering the fructan amounts in plant materialof patent application EP 1084624 A2 is that while Lactobacillus strainsin general do not degrade fructan, there are Lactobacillus strains thatdo have this property. According to EP 1084624 A2, those strains arepreferably Lactobacillus paracasei and Lactobacillus plantarum.

Müller et al (1994) studied fermentation of fructans by epiphytic lacticacid bacteria. Strains of epiphytic lactic acid bacteria were isolatedfrom forage grasses and their ability to hydrolyze fructans was studied.Only 16 out of 712 strains utilized fructans. Said strains wereidentified as Lactobacillus paracasei subsp. paracasei, Lactobacillusbrevis and Pediococcus pentosaceus.

As can be noted from above, some techniques to alter fructan levels arecurrently known and used. Additionally, it is known that sour bread hasnaturally lower levels of fructan. These fructan lowering techniques aregenerally based on using fermentation or specific fructan degradingenzymes.

Several fructan degrading enzymes are known in the art. Glycosidehydrolase family GH32 contains invertases and also enzymes thathydrolyze fructose containing polysaccharides such as inulinases,exo-inulinases, levanases and β-2,6-fructan 6-levanbiohydrolases,fructan β-(2,1)-fructosidase/1-exohydrolases or fructanβ-(2,6)-fructosidase/6-exohydrolases, as well as enzymes displayingtransglycosylating activities such as sucrose:sucrose1-fructosyltransferases, fructan:fructan 1-fructosyltransferases,sucrose:fructan 6-fructosyltransferases, fructan:fructan6G-fructosyltransferases and levan fructosyltransferases.

Extracellular enzymes such as inulinase that hydrolyze fructans areextracted for example from Aspergillus niger and are commerciallyavailable. These extracellular enzymes are naturally occurring enzymesthat are isolated or extracted from their natural environments. However,these extracellular fructanase enzymes are expensive and difficult toobtain in sufficient amounts and high purity for large-scaleapplications.

For example, Paludan-Müller et al (2002) studied purification andcharacterization of an extracellular fructan β-fructosidase from aLactobacillus pentosus strain isolated from fermented fish. Anextracellular fructanhydrolase from Lactobacillus paracasei ssp.paracasei P 4134 was studied by Müller et al (1997), while Goh et al(2007) characterized a fructan hydrolase from Lactobacillus paracasei1195. Document WO 2010/097416 A1 discloses a recombinant protein withfructanase activity comprising a fragment of a natural occurring proteinderived from lactic acid bacteria such as Lactobacillus.

Moreover, with the use of known fructan-degrading enzymes, such asendo-fructanase, inulinase, or levanase, there is a possibility thatfructo-oligosaccharides (FOS) are formed as degradation products as bythis means not all fructan is converted to fructose. Therefore, there isstill a need for a specific fructan degrading enzyme (fructanase,fructan hydrolase) that is able to decompose fructans efficientlywithout formation of FOS.

FOS are carbohydrates that the human body cannot fully digest and canthus function as prebiotics. There are some positive effects suggestedfor FOS. For example, they may produce substances that stop the growthof harmful, toxic gram-negative and positive bacteria in the intestines.However, according to the currently available scientific evidence FOScan execute some harmful effects. FOS can cause e.g. bloating,flatulence, abdominal and intestinal discomfort, and eructation.Furthermore, people with lactose intolerance were shown to particularlysuffer from these side effects. The reason for these symptoms may bethat FOS are generally gastrointestinally more active than fructanpolymer, since the intestinal microflora ferments them more rapidly.Moreover, fructose can also considered being a FODMAP-compound withpeople having impaired fructose absorption. This is a problem when nocomparable amount of glucose is present in the food item or meal. Thisis because fructose absorption in human body occurs along withglucose-induced uptake system. The excess fructose concentration (vsglucose concentration) is, however, easy to tackle with food recipe ormeal formulations.

What is still needed in the art are grain and vegetable materials thatare substantially free of fructans and FOS and thereby can be used toprepare products that are suitable for low-FODMAP diets. What is alsostill needed in the art is an efficient method and means for fructanremoval from grain and vegetable material that would not result inunfavorable degradation products, especially FOS. Therefore, a methodand means that would enable the efficient removal of fructan would bevery beneficial for the development of food products suitable forlow-FODMAP diet. Consumption of these food products would not causegastrointestinal problems. Said food products could even have a positiveeffect on gastrointestinal health and in that way on general wellbeing.

SUMMARY OF THE INVENTION

The invention is defined by the features of the independent claims. Somespecific embodiments are defined in the dependent claims.

The present invention is based on the finding that a novel enzymeisolated from a strain of Lactobacillus is capable to efficientlydegrade and remove fructan of grain and vegetable materials.

According to a first aspect of the present invention there is provided aDNA construct comprising a nucleotide sequence encoding an extracellularfructanase, wherein said nucleotide sequence comprises the nucleotidesequence shown in SEQ ID No. 1 or a sequence analogous thereto having atleast 96% identity to the nucleotide sequence shown in SEQ ID No. 1.

According to a second aspect of the present invention, there is providedan enzyme exhibiting fructan hydrolase activity which enzyme comprises apolypeptide having an amino acid sequence essentially as shown in SEQID. No. 2.

According to a further aspect of the present invention, there isprovided a recombinant expression vector comprising the above mentionedDNA construct, as well as a cell comprising said recombinant expressionvector.

According to a further aspect of the present invention, there isprovided a method of producing an enzyme exhibiting fructan hydrolaseactivity, the method comprising culturing a cell as defined above underconditions permitting the production of the enzyme, and recovering theenzyme from the culture.

According to a further aspect of the present invention, there isprovided an enzyme exhibiting fructan hydrolase activity, which enzymeis encoded by a DNA construct as defined above or is produced by theabove defined method.

According to a further aspect of the present invention, there isprovided an enzyme preparation for the degradation of fructan, saidpreparation comprising an enzyme according to the present invention.

According to a further aspect of the present invention, there isprovided the use of an enzyme or an enzyme preparation according to theinvention for the degradation of fructan in grain materials or invegetables.

According to a further aspect of the present invention, there isprovided the use of an enzyme or an enzyme preparation according to theinvention for preparation of baked products or low-fructan vegetables.

According to a further aspect of the present invention, there isprovided a premix for baking, comprising an enzyme or an enzymepreparation according to the invention, together with one or moreingredients needed or suitable for baking.

A still further aspect of the invention is an improver for baking,comprising an enzyme or enzyme preparation according to the invention,together with one or more ingredients from the group consisting ofenzymes, wheat gluten, carriers (wheat gluten maltodextrin etc.),emulsifiers, such as but not limited to DATEM, and mono anddiglycerides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the percentage of residual fructan when the ability of theenzyme to degrade two inulins of different length, FOS compounds and ryemeal extract was studied as a function of time. In all reactions theenzyme: substrate ratio was about 2.

FIG. 2 shows the amount of substrate degraded by the enzyme per enzymeactivity unit.

FIG. 3 illustrates the amount of fructose formed.

FIG. 4 shows the results of gel filtration chromatography for inulin(DPav=12) and its degradation products. Samples were taken after areaction time of 30 min, 60 min and 120 min. In all samples, thebackground signal caused by the enzyme has been reducted.

FIG. 5 shows the results of gel filtration chromatography for inulin(DPav=25) and its degradation products. Samples were taken after areaction time of 30 min, 60 min and 120 min. In all samples, thebackground signal caused by the enzyme has been reducted.

FIG. 6 shows fructan concentrations in wheat doughs during two hours ofrising. The figure also shows calculated fructan concentration at thebeginning of rising, based on the measured fructan content of wheatflour.

FIG. 7 illustrates the change in fructan concentrations of rye doughsduring two hours of rising. The FIG. also shows theoretical fructanconcentration at the beginning of rising, with and without taking thefructan of the starter culture into account.

FIG. 8 illustrates fructose concentrations in wheat doughs at thebeginning and at the end of rising.

FIG. 9 illustrates fructose concentrations in rye doughs at thebeginning and at the end of rising.

FIG. 10 shows the outlook of baked bread with and without enzymeaddition. Control bread on the left, enzyme bread on the right.

FIG. 11 shows the percentage of residual fructan when the ability of theenzyme to degrade garlic and Jerusalem artichoke was studied as afunction of time.

DETAILED DESCRIPTION OF THE INVENTION

In the present context, the term “analogous” used to define the DNAconstruct of the invention is understood to include any DNA sequencewhich encodes an enzyme with fructanase activity and which is at least96% homologous or has at least 96% identity to the DNA sequence shown inSEQ ID No. 1. The analogous DNA sequence may, e.g. be isolated fromanother organism or may be one prepared on the basis of the DNA sequenceshown in SEQ ID No. 1, such as by introduction of nucleotidesubstitutions which do not give rise to another amino acid sequence ofthe enzyme. Other examples of possible modifications are insertion ofone or more nucleotides into the sequence, addition of one or morenucleotides at either end of the sequence, or deletion of one or morenucleotides at either end or within the sequence.

The present invention provides a novel enzyme that is able toefficiently degrade fructans. The enzyme was isolated and identifiedfrom a strain of Lactobacillus having fructan degrading activity. Morespecifically, the novel extracellular fructanase producing strain wasidentified as Lactobacillus crispatus. A sample of this microorganismwas deposited at the Deutsche Sammlung von Microorganismen undZellkulturen GmbH (DSM) under accession number DSM 29598.

Lactobacillus strains having fructan degrading activity were isolatedfrom a seed starter generated by back slopping. The seed starter wasprepared from grain material having a low content of damaged starch asdisclosed in co-pending application PCT/FI2016/050011.

In brief, a seed starter was produced by utilizing back slopping. Backslopping means that small quantities of dough from the manufacture of afermented product from a previous batch are used as the inoculum orstarter for the subsequent batch production. In the preparation of theseed starter, grain material having a low content, preferably less than1.0% (on grain basis), of damaged starch was used. Grain material wassoaked in liquid, preferably water, and incubated at 20-50° C. for 4 to72 hours. Next day, a fresh batch of the grain material and liquid,preferably water, was mixed as above and inoculated with 1-10% of thepreviously incubated mixture. This back slopping is carried out severaltimes, preferably at least 3-6 times, and can be continued as long asnecessary.

The outcome of the back slopping started from grain material having alow content of damaged starch was the formation of spontaneousmicroflora that contain microbes that are able to efficiently utilizefructans as a carbohydrate source and quantitatively to consume (andthereby remove) fructans from grain raw material. The adapted microflorahad the ability to hydrolyze fructan and further use the possibledegradation products and metabolites (fructose, FOS, mannitol) forgrowth. The flora may also have transport system for fructans or theirhydrolysis products or metabolites.

From the seed starter prepared as above, bacterial colonies withdifferent morphology (outlook) were isolated to pure cultures. Themicrobes of the colonies were analyzed for their efficiency in removingfructan from grain material by using them as pure culture inoculants inlaboratory fermentations.

One isolate effective in fructan removal was sequenced and identified asLactobacillus crispatus (DSM 29598). A novel enzyme of the invention, anextracellular fructosidase and a member of glycosyl hydrolase family 32,was isolated and identified from said strain. It is expected that a DNAsequence coding for a homologous enzyme, i.e. an analogous DNA sequence,may be derived similarly by screening a strain of another microorganism,preferably a Lactobacillus, isolated from a seed starter prepared asdescribed above. Examples of such Lactobacillus strains include but arenot limited to other strains of Lactobacillus crispatus, as well asstrains of Lactobacillus helveticus, Lactobacillus amylovorus,Lactobacillus ultunensis, Lactobacillus amylolyticus, Lactobacillusamylovorans, Lactobacillus sobrius or Lactobacillus acidophilus.

The enzyme protein isolated from Lactobacillus crispatus was found to be95% identical to corresponding proteins in another Lactobacilluscrispatus, and 94% and 93% identical to corresponding proteins in L.amylovorus. None of these Lactobacillus enzymes are biochemicallycharacterized. A fructan hydrolase in L. paracasei was previouslycharacterized (Goh et al 2007) but is not homologous to the fructanhydrolase of the L. crispatus described here. The protein has apredicted sec-dependent signal peptide (VKA-DT) and is an extracellularprotein.

The novel enzyme of the invention operates over a wide temperature rangeand shows relatively high activity between 30-60° C. Optimum temperaturefor fructan hydrolysis is around 50° C. (100% activity) whereas at 30°C. and 60° C. the activity is 80%. The enzyme shows 60% activity at 65°C. and 50% activity at 20° C. The enzyme operates actively in pH range4-6. It shows maximum activity at pH 5.0 and very high activity (>95%)at pH-values 4.5 and 5.5. At pH-values 4.0 and 6.0 the activity is75-80% of the maximum.

The above mentioned properties show that the enzyme of the presentinvention is stable and active over a wide temperature and pH range.Said properties make the enzyme of the present invention particularlysuitable for use in the preparation of low-fructan grain and vegetablematerials as well as low-fructan grain and vegetable ingredients andproducts that are suitable for example for a low-FODMAP diet.

The DNA construct of the invention is understood to include any DNAsequence which encodes an enzyme with fructanase activity and which hasat least 96%, preferably at least 97%), even more preferably at least98%, and still more preferably at least 99% identity to the DNA sequenceshown in SEQ ID No. 1. Thus, the invention is intended to include anychanges in the fructanase coding region which either lead to the sameamino acid sequence or to an amino acid sequence which, notwithstandingone or more deviations from the original amino acid sequence,corresponds to an enzyme having essentially fructanase activity.

A further aspect of the present invention provides a recombinantexpression vector comprising a DNA construct as defined above. A stillfurther aspect is a host cell transformed with a recombinant expressionvector as defined above. The host cell may be for example a bacterium,such as a strain of Escherichia coli, or a yeast, such as Pichiapastoris.

The invention covers the enzyme irrespective of how it has beenproduced, for example by recombinant DNA technology, chemical synthesis,enzymatic degradation or a combination thereof. Further, the inventionnot only covers the enzyme as such, but also in the form of a fusionprotein or as a protein physically or chemically bound to any substanceand having fructanase activity.

Another aspect of the invention is a method of producing an enzymeexhibiting fructanase activity, comprising the expression in a suitablehost of a DNA as defined herein which encodes a fructanase enzyme. Asstated above, the expression may take place in various host cells, amongwhich Pichia pastoris is preferred. The invention also includes a methodas defined above wherein the fructanase enzyme produced is recoveredfrom the culture medium.

An object of the invention is also an enzyme preparation or an improvercomprising an enzyme according to the invention, together with carriersand/or emulsifiers. Carriers may include for example wheat gluten,maltodextrin etc. Suitable emulsifiers include emulsifiers known to aperson skilled in the art, such as for example DATEM.

The enzyme preparation may be prepared in accordance with the methodsknown in the art and may be in the form of a liquid or a drypreparation. For instance, the enzyme preparation may be in the form ofa granulate or a microgranulate. The enzyme to be included in thepreparation may be stabilized in accordance with methods known in theart.

In addition to the enzyme according to the invention, the enzymepreparation may also comprise one or more enzymes having hydrolaseactivity. The enzymes having hydrolase activity may liberate for exampleglucose or maltose. Such enzymes include for example α-glucosidase(liberating glucose from starch/maltodextrin), β-glucosidase (liberatingglucose from β-glucan), invertase (liberating glucose from sucrose), andamylolytic enzymes (liberating maltose from starch). A benefit of havingglucose and fructose present in the product formulation is that fructoseabsorption from small intestine is improved when glucose is present inequal or higher amounts compared with fructose. Another benefit is togain balanced taste of sweetness; although fructose is sweeter thanglucose or maltose or sucrose, its combination with glucose, forinstance, creates sweetness that is perceived more complete in itsprofile of sweet taste. The liberation of sugars (glucose, fructose,maltose) from raw materials or ingredients during food processing, thus,increases “natural”sweetness and, thereby, reduces the need to includeadded sugar in the product formulations.

The novel enzyme and the novel enzyme preparation according to theinvention can be used in the degradation of fructan of grain materialsor vegetables. Suitable grain materials include, without limitation,wheat, rye, barley, and mixtures thereof. A mixture may comprise allthree of the mentioned grain materials or a combination of any two ofthem. Suitable vegetable materials include all vegetables that containfructan (e.g. inulin). Examples of such vegetables include onions,garlic, Jerusalem artichoke, chicory root etc.

The dosage of the novel enzyme and the novel enzyme preparation neededto degrade fructan in a certain material depends on the activity ofenzyme, the amount of fructan in the material and the conditions underwhich the enzyme or the preparation is used and may be determined on thebasis of methods known in the art. For example, at an activity level ofapproximately 500 U/g, an enzyme/substrate ratio of in the range of 2:1to 3:1, preferably about 2.5:1, may be used. In baking, the amount ofenzyme needed naturally depends on the amount of fructan in the floursused. In the case of wheat, 0.1% enzyme based on the weight of wheatflour may be sufficient. In the case of rye, the amount of the enzyme ispreferably over 0.5%, based on the weight of rye flour.

The novel enzyme can thus be used in the preparation of baked products,wherein it has been found to effectively reduce the content of fructan.In laboratory, FOS compounds were almost totally degraded by the enzyme.In rye extract, over 70 or 80% of the fructan of rye extract wasdegraded by the novel enzyme.

When used in baking, the novel enzyme reduced the fructan conten of ryeand wheat doughs to almost zero at the end of rising the dough. At thesame time, the amount of fructose increased compared to a control doughwithout the enzyme.

The novel enzyme can also be used in the preparation low-fructanvegetables wherein it has been found to degrade approximately 60% ormore of the original fructan content of vegetables.

With the novel enzyme or the novel enzyme preparation it is thuspossible to provide wheat, rye, barley and vegetable materials andproducts that are substantially free from fructan and thereby aresuitable for a specific diet such as low-FODMAP diet.

A further object of the invention is a premix for baking comprising thenovel enzyme according to the invention or the enzyme preparationaccording to the invention. Without limitation, premixes typicallyinclude whole, crushed or milled wheat, other cereals, pulses, nuts andseeds, but also carriers, fibers and water binders such as, but notlimited to, maltodextrins, celluloses, pectins, protein concentrates(gluten etc). Premixes may or may not include bread/dough improversand/or their constituents.

A still further aspect of the invention is an improver for baking,comprising an enzyme or enzyme preparation according to the invention,together with one or more ingredients from the group consisting ofenzymes, wheat gluten, carriers (wheat gluten maltodextrin etc.),emulsifiers such as but not limited to DATEM, and mono and diglycerides.

The enzyme may also be used to liberate fructose from fructan. It can beused together with other enzymes, for instance hydrolases that liberateglucose or maltose from sucrose, glucans as starch or beta-glucan ormaltodextrin. Enzymes that can release glucose from described substratesinclude invertases, amylolytic enzymes, alpha-glucosidases andbeta-glucosidases. Release of fructose and/or glucose enables todecrease the amount of added sugar needed to provide the desiredsweetness to the product in question.

At least some embodiments of the present invention find industrialapplication in food industry, in particular in baking products and inpreparation of low FODMAP vegetables. In addition, specific liberationof fructose finds industrial application in food industry as well.

The enzyme of the invention can obviously also be applied outside foodindustry. For instance, the enzyme can be used in biofuel production toliberate fructose from materials containing fructan or in feedproduction to pretreat animal foods to decrease the amount of fructanand thereby to improve the digestion and nutritional value of feed. Forinstance, horses may suffer from laminitis, which is proposed to be thecause of feed containing grains or grass high in non-absorbablecarbohydrates e.g. fructan. This leads to excess gut fermentations whichare believed to cause the condition. The enzyme can also be applied as adigestive-aid enzyme in nutraceutical products similarly as lactaseenzyme is added to improve lactose digestion. The enzyme could also beused in dental care to decrease the amount of plaque. It is known thatfructans play a role in the formation of dental plaque biofilm and thatthe use of fructanase could reduce the amount of plaque.

While the following examples are illustrative of the principles of thepresent invention in one or more particular application, it will beapparent to those of ordinary skill in the art that numerousmodifications in form, usage and details of implementation can be madewithout the exercise of inventive faculty, and without departing fromthe principles and concepts of the invention. Accordingly, it is notintended that the invention be limited, except as by the claims setforth below.

The verbs “to comprise” and “to include” are used in this document asopen limitations that neither exclude nor require the existence of alsounrecited features. The features recited in depending claims aremutually freely combinable unless otherwise explicitly stated.Furthermore, it is to be understood that the use of “a” of “an”, thatis, a singular form, throughout this document does not exclude aplurality.

EXPERIMENTAL Example 1 Production of a Seed Starter and Isolation ofPure Cultures

A seed starter was produced from cut kernels of rye without apre-existing seed starter. The cut kernels used in the example contain0.2% of damaged starch.

100 g of cut kernels were soaked in 150 g of water and incubated at 45°C. After 24 h 10 g of the above mixture was mixed with 100 g of cutkernels of rye and 150 g of water and incubated at 45° C. for 24 h. Thisback slopping was repeated five more times.

From the seed starter prepared as above, bacterial colonies withdifferent morphology (outlook) were isolated to pure cultures. Themicrobes of the colonies were analyzed for their efficiency in removingfructan from grain material by using them as pure culture inoculants inlaboratory fermentations. In each fermentation reaction, 20 g of cutgrains of rye were mixed with 30 grams of tap water and 500 mg pureculture starter suspension containing 10⁹ cells of microbe isolate.After 16 hours fermentation at 37° C., the fructan content of themixtures were analyzed using a commercial kit (K-FRUC, Megazyme). Theinitial fructan content of the grain material was 5% (on a dry matterbasis).

One isolate effective in fructan removal was identified as Lactobacilluscrispatus.

Example 2 Identification of Fructan Hydrolase from L. crispatus (DSM29598)

Genomic DNA isolation and sequencing: Genomic DNA was isolated using theWizard® Genomic DNA Purification Kit (Promega) following themanufacturer's guidelines. The quality and quantity of each sample wasassessed using gel electrophoresis and a NanoDrop Spectrophotometer.Samples were sent to Axeq Technologies (Seoul, South Korea) where theyunderwent further quality checks and genomic sequencing.

Whole-genome sequencing, assembly and annotation: The samples weresequenced using Illumina HiSeq2000 with >500 fold coverage and thequality of the paired-end reads was assessed using the FastQC toolprovided in a Galaxy software bundle. Reads were assembled de novo usingABySS (Assembly By Short Sequence; into contigs using a k-mer value of63. Repetitive sequences and short assemblies were removed by filteringout contigs <500 bp in size. The sequence result was 103 contigs.

The evolutionary history was inferred by using the Maximum Likelihoodmethod based on the JTT matrix-based model. The tree is drawn to scale,with branch lengths measured in the number of substitutions per site.Evolutionary analyses were conducted in MEGA6.

As a result, an extracellular fructosidase, a member of glycosylhydrolase family 32 was identified. The enzyme protein is >93% identicalto corresponding proteins in L. amylovorus, 56% identical to the fructanhydrolase in Atobobium parvulum and >55% identical to fructan hydrolasesin S. mutans. The protein has a predicted sec-dependent signal peptide(VKA-DT) and is thus likely an extracellular protein. The phylogeneticanalysis showed that there are few homologues in Lactobacillus spp.,none of these are biochemically characterized. The fructan hydrolase inL. paracasei that was previously characterized is not homologous to thefructan hydrolase of the presently studied L. crispatus.

Example 3 Production of Enzyme

The enzyme was produced in Pichia pastoris, a methylotrophic yeast thatis widely used in the extracellular expression of recombinant proteins,by following routine recombinant DNA procedures. Standard methods in theenzyme production were performed essentially as described in Maniatis1989, Molecular Cloning, CSH, N.Y., USA.

Example 4 Enzyme Reactions

Fructan Degradation

Fructan degradation ability of the enzyme was examined by two inulins ofdifferent length (inulin HP (DPav=25) and inulin GR (DPav=12)), by FOScompounds and by rye meal extract. For the enzyme reaction, an enzymesolution was prepared, having a concentration of 10 mg/ml, and asubstrate solution, having a concentration of 4 mg/ml. For themanufacture of both solutions, 0.1 M sodium acetate buffer (pH 4.5) wasused. 0.5 ml of each solution was transferred to the reaction mixture.When rye extract was used as a substrate, the concentrations were halfthe size. The enzyme reaction was carried out at 50° C. and the reactiontime was 2 hours. Samples were taken after 60 min and after 120 min. Asolution wherein the enzyme solution was replaced by 0.5 ml of 0.1 Msodium acetate buffer (pH 4.5) was used as a standard. Reactions werestopped by placing the samples for 5 min in a boiling water bath, andthen they were allowed to stand for 5 min in a cold water bath. Prior tothe determination of fructan concentrations, the samples were allowed tostand for about 10 min at room temperature. They were diluted withdeionized water so that the maximum fructan concentration was 1 mg/ml.The amount of fructan degradation by the enzyme was obtained bysubtracting the amount of fructan in the reaction mixture from theamount of fructan in the standard.

In the reactions an enzyme compound was used, which had declaredactivity of 516.6 U/g. The enzyme/substrate ratio in the reactions wasabout 2. FIG. 1 shows the percentage of remaining residual fructan basedon the initial concentration. The diagram shows that the FOS compoundsare mostly degraded by the enzyme, while longer chain inulin is lessdegraded. After two hours, 68.5% of the longer inulin was remainingwhereas there were only 3.0%) of FOS compounds. The shorter inulin andrye extract were about equally broken down in percentage terms (inulin25.3% and rye extract 27.1% fructan left).

Since the enzyme/substrate ratios varied slightly for each substrate,the amount of substrate degraded by the enzyme was calculated also perenzyme activity unit (FIG. 2). The highest degradation was found withFOS compounds, while the shorter inulin had the lowest degradation.During two hours, FOS compounds had a degradation rate of 0.654 mg/U,whereas that of inulin was 0.25 mg/U. Fructan of rye extract had abetter degradation rate relative to the amount of enzyme than theshorter inulin (rye 0.598 mg/U and inulin 0.541 mg/U).

Both diagrams show that the enzyme decomposes substrates at a higherrate during the first hour and thereafter the degradation ratedecreases.

Fructose Formation

In addition to fructan concentrations, also fructose concentrations inthe reactions were measured after 60 min and 120 min reaction times.FIG. 3 shows the increase in fructose concentrations as a function oftime. The resulting fructose is shown per enzyme activity unit. Thehighest amounts of fructose were formed with rye extract and FOScompounds as substrates. From the shorter inulin 0.775 mg U fructose wasformed in two hours. The lowest amount of fructose was formed when thelonger inulin was the substrate (0.431 mg/U). When rye extract and FOScompounds were used as substrates, the formation of fructose wassignificantly lower after an hour. Fructose formation from inulin wasalmost at the same level during the two hours. The samples were alsoassayed for glucose and sucrose content, but those compounds were almostnot formed in the reactions.

Degrees of hydrolysis were calculated on the basis of fructose formationof different substrates. FOS compounds hydrolyzed almost completely(96.6%), which was very close to the value obtained from fructan assays.Also, degrees of hydrolysis of the shorter inulin and rye extract werevery close to the estimated degradation rate based on fructanmeasurements. Instead, longer inulin hydrolyzed considerably morecalculated on the basis fructose concentrations, the degree ofhydrolysis being 55.0%>, based on fructose, and 31.5%, based on fructan.

TABLE 8 Calculated degrees of hydrolysis based on fructose formation forvarious substrates after a reaction time of two hours Substrate Degreeof hydrolysis (%) FOS 96.6 Inulin (DPav = 10) 75.5 Inulin (DPav = 23)55.0 Rye extract 81.3

Hydrolysis Method

Gel filtration chromatography was used to clarify whether the enzymefirst degrades its substrate into FOS compounds or whether it releasessingle fructose molecules from the ends of fructan chain. Measurementswere made for inulin reactions and the degradation products weredetermined for samples taken at 30 min, 60 min and 120 min. In addition,blank samples containing only the substrate were determined. FIG. 4shows the spike formed by the shorter inulin and its degradationproducts. The low peak at about 50 minutes represents inulin and theother peaks represent degradation products. The highest peak on thegraph shows the fructose, and FOS compounds are also formed in thereaction. Based on the molecular weights, FOS compounds have about 2 to3 fructose units.

FIG. 5 shows the peak formed by the longer inulin and its degradationproducts. The reaction forms the same end products as the reaction ofthe shorter inulin. However, at the site of FOS compounds there are moreof those FOS compounds that are three units long insulin left even aftertwo hours.

Example 5 Baking

The ability of the enzyme to degrade fructan in a baking process wasstudied by adding enzyme to wheat and rye doughs. Table 1 shows thebasic recipe of wheat and rye doughs. The amount of enzyme in the wheatdough was 0.175%, based on the weight of wheat flour, and in rye dough0.68%, based on the weight of rye flour. The flour in the starterculture is not taken into account in the calculation. In addition toenzyme doughs/breads, control doughs without added enzyme were prepared.

TABLE 1 Ingredients of wheat and rye doughs and their amounts based onthe amounts of wheat and rye flour Wheat dough Rye dough Ingredient (%)(%) Wheat flour 100 — Rye flour — 100 Starter culture — 74 Water 67 61Yeast 3 1.2 Salt 1.7 2.3 Sugar 1.8 — Oil 1.2 —

The doughs were prepared by mixing all the ingredients. Wheat doughswere stirred for approximately 5 min and rye doughs until theingredients were mixed. The enzyme was added to water before the otheringredients. The doughs were allowed to rise for two hours at atemperature of 37° C. Samples of the doughs were taken immediately aftermixing and after rising of 30 min, 60 min and at the end of the rising.The doughs were baked for 20 min at a temperature of 210° C. The lastsample was taken from cooled breads. All the samples were frozen andassayed for fructan and fructose concentrations. Rye dough samples wereassayed also for mannitol concentrations.

Fructan Concentrations in Doughs

Fructan concentrations in wheat doughs during two hours of rising areshown in FIG. 6. Fructan concentration in the control dough was 0.43% atthe beginning of rising and it decreased to 0.23% during two hours. Inthe dough with added enzyme, the concentration was 0.37%) at thebeginning of rising and 0.04% at the end of rising. In both doughs, thefructan concentrations were lower than was expected based on the fructanconcentration of wheat flour.

FIG. 7 shows the change in fructan concentrations of rye doughs duringtwo hours of rising. FIG. 7 also shows theoretical fructanconcentrations at the beginning of rising, with and without taking thefructan of the starter culture into account. Fructan concentration inthe control dough was 1.6% at the beginning of rising and 1.3% at theend of rising. In the dough with added enzyme, the concentrations were1.0% at the beginning and 0.08%) at the end. The diagram also shows howthe fructan content of the enzyme dough is remarkably smaller than thatof the control already at the beginning of rising.

Fructose Concentrations in Doughs

Fructose concentrations in the doughs were determined at the beginningand at the end of rising. FIG. 8 shows the changes in fructoseconcentrations of the wheat doughs after two hours. Surprisingly, thefructose content of the control dough (0.64%) at the beginning of risingwas higher than that of the enzyme dough (0.54%). During two hours,however, the control dough concentration decreased considerably morethan that of the enzyme dough, the concentrations being 0.17% in thecontrol dough and 0.38% in the enzyme dough.

In rye doughs, the fructose concentrations remained also the same whenconcentrations at the beginning and at the end of rising were compared(FIG. 9). The fructose concentrations of the control dough were 0.30% atthe beginning and 0.32% at the end of rising. In the enzyme dough,fructose concentration was 1.29% at the beginning and 1.39% at the endof rising.

Baked Bread

Finally, the doughs were baked and fructan and fructose concentrationsof the final baked breads were determined. Mannitol concentrations ofrye bread were also determined. Mannitol assay was made byD-mannitol/L-arabitol Assay Kit method. 1-2 g samples were weighed andthen they were dissolved in water by heating and stirring. The assay wasmade according to the instructions of the manufacturer. In the treatmentof solid samples, the samples were not filtered after dissolving inwater but centrifuged for 5 minutes (5000 rpm).

The results are summarized in Table 2. Overall, the levels increasedslightly during cooking (water evaporation). Fructan content of wheatbread containing enzyme was 0.05%). Fructan content of rye breadcontaining enzyme was 0.15%. Mannitol concentrations of the rye breadswere very similar to each other. In particular, rye bread with enzymecontained more fructose than the control.

TABLE 2 Fructan, fructose and mannitol concentrations of baked breadsFructan Fructose Mannitol Bread (%) (%) (%) Wheat, control 0.25 0.01 —Wheat, enzyme 0.05 0.40 — Rye, control 1.40 0.20 0.34 Rye, enzyme 0.151.31 0.36

Baked wheat and rye breads were also sensory evaluated. Evaluatedproperties included crust color, texture and softness of the bread, aswell as shelf life. The breads were also weighed and measured forvolume. There was hardly any difference in the enzyme breads compared tothe control breads. The only difference detected was the crust colour ofwheat breads. The crust of the enzyme bread was slightly darker thanthat of the control (FIG. 11).

Example 6 Vegetable Treatment

a) Garlic

Garlic contained around 20 g fructan/100 g (fresh weight). Four grams ofgarlic was crushed and mixed with 50 mL of tap water. The fructanaseenzyme (1000 U) was added and the suspension was incubated at 50° C. for5 hours. Samples were analysed at time points of 0 h, 4 h, and 5 h. Thefructan content decreased during 4-5 hours of incubation leaving theresidual fructan content 40% of the 0 h sample (FIG. 11).

b) Jerusalem artichoke

Jerusalem artichoke contained around 12% fructan/100 g (fresh weight).Eight grams of Jerusalem artichoke was sliced cut into small pieces andmixed with 30 mL of tap water. The fructanase enzyme (1250 U) was addedand the mixture was incubated at 50° C. for 5 hours. Samples wereanalysed at time points 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h. The fructancontent decreased steadily leaving 32% of residual fructan after 5h ofincubation (FIG. 11).

Example 7 Baking (mixture of flours)

The enzyme was tested in a straight dough baking process for mix-breadcontaining a mixture of wheat, oats and rye flours. Ingredients (seeTable 3) were mixed for 5 min in a dough mixer and the dough was allowedto rest for 20 min.

TABLE 3 Ingredients of mix-bread containing wheat, oats and rye flours(g) Ingredient Blanco Enzyme Water 2.000 2.000 Wheat flour 0.800 0.800Oat flour 0.800 0.800 Rye flour 0.500 0.500 Salt 0.050 0.050 Oil 0.1000.100 Dry yeast 0.020 0.020 Enzyme 0 0.004

The dough was moulded to flat breads that were proofed for 45 min at 40°C. and oven-baked at 230° C. for 15min and cooled down at roomtemperature. The fructan content was determined after cooling. Thefructan contents were 0.34% for Bianco-bread and 0.17% for Enzyme-bread.

CITATION LIST Patent literature

EP 1084624 A2

US 20110129572 A1

WO 2010/097416 A1

Non Patent Literature

Andersson, R., Fransson, G., Tietjen, M. & Åman, P. (2009). Content andmolecular

weight distribution of dietary fiber components in whole-grain rye flourand bread. Journal of Agricultural and Food Chemistry 57 (5), 2004-2008.

Goh Y J., Lee J H & Hutkins R W. (2007) Functional Analysis of theFructooligosaccharide Utilization Operon in Lactobacillus paracasei1195. Appl. Environ. Microbiol. 73 (18) 5716-5724.

Müller, M. and Lier, D. (1994). Fermentation of fructans by epiphyticlactic acid bacteria. Journal of Applied Bacteriology 76 (4), 406-411.

Müller, M. and Seyfarth, W. (1997). Purification and substratespecificity of an extracellular fructanhydrolase from Lactobacillusparacasei ssp. paracasei P 4134. New Phytol. 136, 89-96.

Paludan-Müller, C., Gram L. & Rattray, F. P. (2002). Purification andCharacterisation of an Extracellular Fructan β-fructosidase from aLactobacillus pentosus Strain isolated from Fermented Fish. System.Appl. Microbiol. 25, 13-20.

Rakha A., Åman, P. & Andersson, R. (2010). Characterisation of dietaryfibre components in rye products. Food Chemistry 119 (3), 859-867.

1. A DNA construct comprising a nucleotide sequence encoding anextracellular fructosidase, wherein said nucleotide sequence comprisesthe nucleotide sequence shown in SEQ ID No. 1 or an analogous sequencethereof having at least 96% identity to the nucleotide sequence shown inSEQ ID No.
 1. 2. The DNA construct according to claim 1, wherein thenucleotide sequence encoding an extracellular fructosidase is obtainablefrom a strain of Lactobacillus.
 3. The DNA construct according to claim2, wherein the nucleotide sequence is obtainable from a strain ofLactobacillus, in particular a strain of Lactobacillus crispatus,Lactobacillus helveticus, Lactobacillus amylovorus, Lactobacillusultunensis, Lactobacillus amylolyticus, Lactobacillus amylovorans,Lactobacillus sobrius or Lactobacillus acidophilus.
 4. The DNA constructaccording to claim 3, wherein the nucleotide sequence is obtainable fromLactobacillus crispatus (DSM 29598).
 5. A recombinant expression vectorcomprising a DNA construct, wherein the DNA construct comprises anucleotide sequence encoding an extracellular fructosidase, wherein saidnucleotide sequence comprises the nucleotide sequence shown in SEQ IDNo. 1 or an analogous sequence thereof having at least 96% identity tothe nucleotide sequence shown in SEQ ID No
 1. 6. A cell comprising arecombinant expression vector, wherein the recombinant expression vectorcomprises a DNA construct and .wherein the DNA construct comprises anucleotide sequence encoding an extracellular fructosidase wherein saidnucleotide sequence comprises the nucleotide sequence shown in SEQ IDNo. 1 or an analogous sequence thereof having at least 96% identity tothe nucleotide sequence shown in SEQ ID No.
 1. 7. A method of producingan enzyme exhibiting fructan hydrolase activity, the method comprisingculturing a cell a under conditions permitting the production of theenzyme, and recovering the enzyme from the culture, wherein the cellcomprises a recombinant expression vector, wherein the recombinantexpression vector comprises a DNA construct, and wherein the DNAconstruct comprises a nucleotide sequence encoding an extracellularfructosidase, wherein said nucleotide sequence comprises the nucleotidesequence shown in SEQ ID No. 1 or an analogous sequence thereof havingat least 96% identity to the nucleotide sequence shown In SEQ ID No. 1.8. An enzyme exhibiting fructan hydrolase activity, wherein the enzymeis encoded by a DNA construct or produced by a method of producing anenzyme exhibiting fructan hydrolase activity, wherein the DNA constructcomprises a nucleotide sequence encoding an extracellular fructosidase,wherein said nucleotide sequence comprises the nucleotide sequence shownin SEQ ID No. 1 or an analogous sequence thereof having at least 96%identity to the nucleotide sequence shown in SEQ ID No. 1, and whereinthe method of producing an enzyme exhibiting fructan hydrolase activitycomprises culturing a ceil tinder conditions permitting the productionof the enzyme, and recovering the enzyme from the culture, wherein thecell comprises a recombinant expression vector, wherein the recombinantexpression vector comprises a DNA construct. and wherein the DNAconstruct comprises a nucleotide sequence encoding an extracellularfructosidase, wherein said nucleotide sequence comprises the nucleotidesequence shown in SEQ ID No. 1 or an analogous sequence thereof havingat least 96% identity to the nucleotide sequence shown in SEQ ID No. 1.9. An enzyme exhibiting fructan hydrolase activity, which enzymecomprises a polypeptide having an amino acid sequence essentially asshown in SEQ ID. No.
 2. 10. An enzyme preparation for the degradation offructan, said preparation comprising an enzyme exhibiting fructanhydrolase activity, together with carriers and/or emulsifiers, whereinthe enzyme is encoded by a DNA construct or produced by a method ofproducing an enzyme exhibiting fructan hydrolase activity, wherein theDNA construct comprises a nucleotide sequence encoding an extracellularfructosidase, wherein said nucleotide sequence comprises the nucleotidesequence shown in SEQ ID No. 1 or an analogous sequence thereof havingat least 96% identity to the nucleotide sequence shown in SEQ ID 1, andwherein the method of producing an enzyme exhibiting fructan hydrolaseactivity comprises culturing a cell under conditions permitting theproduction of the enzyme, and recovering the enzyme from the culture,wherein the ceil comprises a recombinant expression vector, wherein therecombinant expression vector comprises a DNA construct, and wherein theDNA construct comprises a nucleotide sequence encoding an extracellularfructosidase, wherein said nucleotide sequence comprises the nucleotidesequence shown in SEQ ID No. 1 or an analogous sequence thereof havingat least 96% identity to the nucleotide sequence shown in SEQ ID No. 1.11. The enzyme preparation according to claim 10, which additionallycomprises one or more of other enzymes having hydrolase activity. 12.The enzyme preparation according to claim 11, wherein the otherenzyme(s) is/are liberating glucose or maltose.
 13. The enzymepreparation according to claim 12, wherein glucose is liberated fromstarch/maltodextrin by α-glucosidase.
 14. The enzyme preparationaccording to claim 12, wherein glucose is liberated from b-glucan byβ-glucosidase.
 15. The enzyme preparation according to claim 12, whereinglucose is liberated from sucrose by an invertase.
 16. The enzymepreparation according to claim 12, wherein maltose is liberated fromstarch by amylolytic enzymes.
 17. Use of an enzyme exhibiting fructanhydrolase activity or an enzyme preparation for the degradation offructan in grain materials or in vegetables, wherein the enzyme isencoded by a DNA construct, or produced by a method of producing anenzyme exhibiting fructan hydrolase activity. wherein the DNA constructcomprises a nucleotide sequence encoding an extracellular fructosidase,wherein said nucleotide sequence comprises the nucleotide sequence shownin SEQ ID No. 1 or an analogous sequence thereof having at least 96%identity to the nucleotide sequence shown in SEQ ID No. 1, and whereinthe method of producing an enzyme exhibiting fructan hydrolase activitycomprises culturing a cell under conditions permitting the production ofthe enzyme. and recovering the enzyme from the culture, wherein the ceilcomprises a recombinant expression vector, wherein the recombinantexpression vector comprises a DNA construct and wherein the DNAconstruct comprises a nucleotide sequence encoding an extracellularfructosidase, wherein said nucleotide sequence comprises the nucleotidesequence shown in SEQ ID No. 1 or an analogous sequence thereof havingat least 96% identity to the nucleotide sequence shown in SEQ ID No 1.wherein the enzyme preparation comprises an enzyme exhibiting fructanhydrolase activity, together with carriers and/or emulsifiers, whereinthe enzyme is encoded by a DNA construct or produced by a method ofproducing an enzyme exhibiting fructan hydrolase activity, wherein theDNA construct comprises a nucleotide sequence encoding an extracellularfructosidase. wherein said nucleotide sequence comprises the nucleotidesequence shown in SEQ ID No. 1 or an analogous sequence thereof havingat least 96% identity to the nucleotide sequence shown in SEQ ID No 1,and wherein the method of producing an enzyme exhibiting fructanhydrolase activity comprises culturing a cell under conditionspermitting the production of the enzyme, and recovering the enzyme fromthe culture wherein the cell comprises a recombinant expression vector,wherein the recombinant expression vector comprises a DNA construct andwherein the DNA construct comprises a nucleotide sequence encoding anextracellular fructosidase, wherein said nucleotide sequence comprisesthe nucleotide sequence shown in SEQ ID No. 1 or an analogous sequencethereof having at least 96% identity to the nucleotide sequence shown inSEQ ID No.
 1. 18. Use of an enzyme or an enzyme preparation for thepreparation of baked products. wherein the enzyme is encoded by a DNAconstruct or produced by a method of producing an enzyme exhibitingfructan hydrolase activity, wherein the DNA construct comprises anucleotide sequence encoding an extracellular fructosidase, wherein saidnucleotide sequence comprises the nucleotide sequence shown in SEQ IDNo. 1 or an analogous sequence thereof having at least 96% identity tothe nucleotide sequence shown in SEQ ID No. 1, and wherein the method ofproducing an enzyme exhibiting fructan hydrolase activity comprisesculturing a cell under conditions pertaining the production of theenzyme, and recovering the enzyme from the culture, wherein the cellcomprises a recombinant expression vector, wherein the recombinantexpression vector comprises a DNA construct and wherein the DNAconstruct comprises a nucleotide sequence encoding an extracellularfructosidase, wherein said nucleotide sequence comprises the nucleotidesequence shown in SEQ ID No. 1 or an analogous sequence thereof havingat least 96% identity to the nucleotide sequence shown in SEQ ID No. 1,wherein the enzyme preparation comprises an enzyme exhibiting fructanhydrolase activity, together with carriers and/or emulsifiers, whereinthe enzyme is encoded by a DNA construct or produced by a method ofproducing an enzyme exhibiting fructan hydrolase activity, wherein theDNA construct comprises a nucleotide sequence encoding an extracellularfructosidase, wherein said nucleotide sequence comprises the nucleotidesequence shown in SEQ ID No. 1 or an analogous sequence thereof havingat least 96% identity to the nucleotide sequence shown in SEQ ID No. 1,and wherein the method of producing an enzyme exhibiting fructanhydrolase activity comprises culturing a cell under conditionspermitting the production of the enzyme, and recovering the enzyme fromthe culture, wherein the cell comprises a recombinant expression vector,wherein the recombinant expression vector comprises a DNA construct, andwherein the DNA construct comprises a nucleotide sequence encoding anextracellular fructosidase, wherein said nucleotide sequence comprisesthe nucleotide sequence shown in SEQ ID No. 1 or an analogous sequencethereof having at least 96% identity to the nucleotide sequence shown inSEQ ID No.
 1. 19. (canceled)
 20. The use according to claim 17, whereinthe grain material is selected from the group consisting of wheat, rye,barley, and mixtures thereof and the vegetables are selected from thegroup consisting of onion, garlic, and Jerusalem artichoke.
 21. A premixor improver for baking, comprising an enzyme or an enzyme preparationwherein the enzyme is encoded by a DNA construct or produced by a methodof producing an enzyme exhibiting fructan hydrolase activity, whereinthe DNA construct comprises a nucleotide sequence encoding anextracellular fructosidase, wherein said nucleotide sequence comprisesthe nucleotide sequence shown in SEQ ID No. 1 or an analogous sequencethereof having at least 96% identity to the nucleotide sequence shown inSEQ ID No. 1, and wherein the method of producing an enzyme exhibitingfructan hydrolase activity comprises culturing a cell under conditionspermitting the production of the enzyme, and recovering the enzyme fromthe culture, wherein the cell comprises a recombinant expression vector,wherein the recombinant expression vector comprises a DNA construct andwherein the DNA construct comprises a nucleotide sequence encoding anextracellular fructosidase, wherein said nucleotide sequence comprisesthe nucleotide sequence shown in SEQ ID No. 1 or an analogous sequencethereof having at least 96% identity to the nucleotide sequence shown inSEQ ID No. 1, wherein the enzyme preparation comprises an enzymeexhibiting fructan hydrolase activity, together with earners and/oremulsifiers, wherein, the enzyme is encoded by DNA construct or producedby a method of producing an enzyme exhibiting fructan hydrolaseactivity, wherein the DNA construct comprises a nucleotide sequenceencoding an extracellular fructosidase. wherein said nucleotide sequencecomprises the nucleotide sequence shown in SEQ ID No. 1 or an analogoussequence thereof having at least 96% identity to the nucleotide sequenceshown in SEQ ID No. 1, and wherein the method of producing an enzymeexhibiting fructan hydrolase activity comprises culturing a cell underconditions permitting the production of the enzyme, and recovering theenzyme from the culture, wherein the cell comprises a recombinantexpression vector., wherein the recombinant expression vector comprisesa DNA construct, and wherein the DNA construct comprises a nucleotidesequence encoding an extracellular fructosidase, wherein said nucleotidesequence comprises the nucleotide sequence shown in SEQ ID No. 1 or ananalogous sequence thereof having at least 96% identity to thenucleotide sequence shown in SEQ ID No.
 1. 22. The premix according toclaim 21, further comprising ingredients selected from the groupconsisting of whole, crushed and milled wheat, other cereals, pulses,nuts, seeds, carriers, fibers, and water binders such as, but notlimited to, maltodextrins, celluloses, pectins, protein concentrates(gluten etc), bread/dough improvers and/or their constituents. 23.(canceled)